PERIOD 2 regulates low-dose radioprotection via PER2/pGSK3β/β-catenin/Per2 loop

Summary During evolution, humans are acclimatized to the stresses of natural radiation and circadian rhythmicity. Radiosensitivity of mammalian cells varies in the circadian period and adaptive radioprotection can be induced by pre-exposure to low-level radiation (LDR). It is unclear, however, if clock proteins participate in signaling LDR radioprotection. Herein, we demonstrate that radiosensitivity is increased in mice with the deficient Period 2 gene (Per2def) due to impaired DNA repair and mitochondrial function in progenitor bone marrow hematopoietic stem cells and monocytes. Per2 induction and radioprotection are also identified in LDR-treated Per2wt mouse cells and in human skin (HK18) and breast (MCF-10A) epithelial cells. LDR-boosted PER2 interacts with pGSK3β(S9) which activates β-catenin and the LEF/TCF mediated gene transcription including Per2 and genes involved in DNA repair and mitochondrial functions. This study demonstrates that PER2 plays an active role in LDR adaptive radioprotection via PER2/pGSK3β/β-catenin/Per2 loop, a potential target for protecting normal cells from radiation injury.


INTRODUCTION
The biological system has developed unique adaptation ability to survive under hostile genotoxic milieu. The background natural low-dose radiation (LDR) and the circadian rhythm, both are evident on the Earth's surface, may coordinatively contribute to the acclimating competence of mammalian cells. It has been long recognized that disturbed circadian rhythm and/or overexposure to ionizing radiation can impair cellular homeostasis leading to aging and risk of many human diseases including cancer generation. [1][2][3][4] Animals with disturbed circadian oscillation or carrying a deficient clock protein are sensitive to radiation-induced cell injury, DNA damage response, and cell transformation potential. [5][6][7][8][9] However, in contrast, mammalian cells exposed to naturally LDR can develop a temporary but significant cellular tolerance to subsequent genotoxic conditions such as a lethal dose of ionizing radiation. [10][11][12][13] Elucidating the specific clock proteins in signaling the LDR-induced radiation tolerance will help to understand an integrated mechanism underlying cellular stress response.
In addition to the natural LDR which includes the radioactive sources in earth, river and atmosphere, epidemiological analysis raises a health concern on the artificial LDR such as medical diagnosis and industrial radiation applications. 14-18 DNA damage response and dynamic mitochondrial metabolism, two fundamental cellular functions in cell response to genotoxic stresses are linked with circadian oscillation. [19][20][21] Dysfunctional regulation of clock genes contributes to cell radiosensitivity and radiation-induced cancer incidence 7 as well as the tumor chemoresistance 22 and radioresistance. 23 It is unclear however if such circadian sensitive radiation injuries can be compromised by LDR-induced cellular homeostasis via clock protein regulation.
The period circadian clock 2 (PER2) is a well-defined clock protein functioning in the regulation of different cellular functions and coordinating with circadian rhythmicity. [24][25][26] PER2 is actively involved in the regulation of cell cycle progression and as a transcriptional regulator, PER2 generates a negative feedback signal to endure the circadian periodicity. 27,28 PER2 is shown to regulate the p53 signaling pathway in DNA damage response. 19 Studies conducted with animal models and epidemiological analyses further demonstrate  iScience Article that loss of PER2 function contributes to dysregulated mitochondrial metabolism in cancer initiation potential. 3 However, how PER2 functions in signaling LDR-induced prosurvival pathways are yet to be identified.
GSK3b is a serine/threonine kinase that plays a significant role in the Wnt/b-catenin signaling pathway. 29 GSK3b regulation is affected by circadian rhythmicity in which active GSK3b phosphorylates and activates PER2 for nuclear translocation 30 and PER2 protein stability is regulated by active b-catenin 31 as well as by circadian regulated cellular redox imbalance. 32 Herein, this study provides the evidence that PER2 plays a critical role in signaling LDR-induced adaptive radioprotection. Per2 gene transcription is first enhanced by LDR and PER2 further upregulates a cluster of prosurvival genes including DNA repair and mitochondrial metabolism via PER2/pGSK3b(S9) interaction leading to active b-catenin nuclear translocation and TCF/ LEF mediated gene transcription. These findings suggest that PER2/pGSK3b(S9) interaction is a potential therapeutic target in protecting normal cells from radiation injury.

RESULTS
Per2 def mice are radiosensitive with impaired DNA repair Although disturbed circadian rhythm is long recognized to raise animal radiosensitivity, it remains unclear if specific clock proteins are decisive in animal survival after radiation. In agreement with the reported radiosensitivity by disturbed circadian rhythm, 6 it was confirmed with mice carrying a deficient form of Per2 (Per2 def ; an in-frame deletion in the PAS-B domain) 7 compared to the counterpart C57BL/C6 Per2 wild type (Per2 wt ) mice. We found that the survival rates were significantly reduced in Per2 def mice following whole-body irradiation (WBI) with doses of 9, 10, and 12 Gy; whereas 7 Gy irradiation showed a less survival effect with no statistical difference was obtained (p = 0.0927, Figure 1A). These results are complementary to the report that except for accelerated aging, no difference was observed in survival and tumor incidence between Per2 def versus Per2 wt mice following WBI with 4 Gy. 33 Thus, a threshold dose level is involved in the radiosensitivity of Per2 def animals. Consistent with the enhanced animal radiosensitivity, DNA damage levels were remarkedly elevated in bone marrow monocytes (BMMNCs) of Per2 def mice compared to Per2 wt mice measured by comet assay and DNA tail moment ( Figures 1B and 1C). In consistence, reduced DNA damage repair ability was detected in the irradiated Per2 def BMMNCs compared to the Per2 wt BMMNCs measured by gH2AX foci with the binding of 53BP1 and Rad51 for non-homologous end-joining (NHEJ) and homologous recombination (HR) repair, respectively ( Figures 1D-1G), indicating that PER2 is involved in cell radiosensitivity via DNA repair ability.

Identification of PER2-related prosurvival genes
RNAseq analysis was then conducted using the lineage bone marrow progenitor hematopoietic stem cells (BMpHSCs) isolated from mice Per2 wt and Per2 def bone marrow cells via sorting Lin À /Sca-1 + /c-Kit + population ( Figure S1). Interestingly, compared to 1.40% population of Lin À /Sca-1 + /c-Kit + BMpHSCs detected in Per2 wt mice 0.86% BMpHSCs were obtained in Per2 def mice ( Figures 2E and S2). In addition, the Per2 def BMMNCs also demonstrated a pro-apoptotic tendency in LDR and clonogenic incapability in both basal and LDR conditions ( Figures 2F, 2G, and S3). These results suggest that PER2 is involved in cellular adaptive radioresistance via the regulation of prosurvival genes.  Mitochondrial functions are tightly associated with nuclear genomic stability and DNA repair, [34][35][36] which endeavored us to assume that genes involved in mitochondrial metabolism could be affected in the Per2 def BMHSCs ( Figure 3A). The top downregulated categories in Per2 def BMHSCs via RNAseq profiling were: cytochrome complex assembly; mitochondrial respiratory chain complex assembly; NADH dehydrogenase complex assembly; mitochondrial respiratory chain complex I assembly and mitochondrial gene expression ( Figure 3B). A group of key mitochondrial metabolism elements including CPT1A, CPT2, NDUFA12, and NDUFV3 was also identified ( Figure 3C) with additional Per2-related mitochondrial metabolic genes illustrated in Figure S4. In consistence with the lack of genes in mitochondrial functions and contrasted with Per2 wt counterpart, Per2 def BMMNCs demonstrated reduced mitochondrial membrane potential ( Figure 3D), oxygen consumption (OC, Figure 3E), and ATP generation ( Figure 3F) without debatable LDR-induced mitochondrial adaptive metabolic activity. Together with the impaired DDR capacity shown in Figure 2, these results suggest that Per2 plays a critical role in the signaling network required for cellular adaptive response to genotoxic stress by involving DNA repair and mitochondrial metabolic functions.

PER2 induction for low-level radiation adaptive radioprotection
To test if Per2 is necessary in LDR-induced adaptive radioprotection, we observed that enhanced Per2 expression was detected in Per2 wt BMMNCs, mouse embryonic fibroblasts (MEFs), human mammary epithelial (MCF-10A), and human skin keratinocytes (HK18) at different times following LDR (Figures 4A and 4B). Per2 was enhanced with a peak time around 8-12 h after LDR in BMMNCs, MCF-10A, and HK18 cells and a consistent raised level starting at 4 h in MEF. In addition, LDR-enhanced Per2 expression was identified in the primary cultured mammary epithelial cells isolated from a healthy woman with breast reduction surgery ( Figure 4C). LDR-mediated radioprotection was recaptured in MCF-10A cells using established protocol 12,37 of LDR before exposure to genotoxic high dose radiation (HDR, 5 Gy) measured by apoptosis and clonogenic survival ( Figures 4D, 4E, and S5A). To further determine that Per2 participates in LDR-induced adaptive radioprotection, Per2 wt and Per2 def BMMNCs were exposed to LDR with or without the challenging HDR. Per2 wt cells showed the adaptive radioprotection with decreased apoptosis and enhanced GM-CFUs comparing to cells without LDR pre-exposure, however it was impaired in Per2 def BMMNCs ( Figures 4F, 4G, and S5B). A specific human Per2 siRNA with 30 nM effectively blocked Per2 expression contrasted with the scrambled control ( Figures S5C and S5D) and eliminated LDR-mediated cellular adaptive radioprotection in MCF-10A cells measured by cell proliferation and clonogenic survival ( Figures 4H and 4I), indicating that PER2 is able to initiate a specific signaling pathway in adaptive radioprotection of cells.
PER2 activates GSK3b/b-catenin pathway with Per2 expression Wnt/b-catenin pathway plays an important role in the regulation of cell proliferation and radiation response. [38][39][40] We found that Per2 expression is positively correlated with GSK3b and b-catenin gene (CTNNB1) in the profile of human breast mammary tissue collected at GEPIA (Figures 5A and 5B). Intriguingly, although both phosphorylated AKT (p-AKTS473), a AKT active form and upstream regulator of GSK3b, and the phosphorylated GSK3b/ser9 (pGSK3bS9) remained unchanged in Per2 wt and Per2 def BMMNCs, active b-catenin was remarkably reduced in Per2 def BMMNCs ( Figure 5C). These results suggest a potential critical mechanism in which PER2 controls the release of b-catenin from the GSK3b/b-catenin complex for regulating a cluster of prosurvival genes. Indeed, like Per2 induction, pGSK3b(S9) was induced iScience Article with a similar peak time (8-12 h) after LDR in Per2 wt /GSKß wt MEFs following exposure to LDR ( Figure 5D) with which LDR-induced adaptive radioprotection was recaptured whereas in Per2 wt /GSK3ß ko MEFs no such adaptive radioprotection was induced ( Figures 5E and S6). In consistence, pGSK3b(S9) was enhanced in Per2 wt BMMNCs but not in Per2 def BMMNCs and the LDR-induced pGSK3b(S9) was accompanied by increased active form of b-catenin in Per2 wt BMMNCs, but not in Per2 def BMMNCs ( Figures 5F and 5G). Since GSK3b mediated the phosphorylation of b-catenin leading to b-catenin degradation 41 we assumed that LDR-enhanced PER2 regulates a prosurvival gene expression via b-catenin activation. As expected, in (C-E) Western blot of Per2 in primary cultured epithelial cells derived from healthy human mammary tissues 12 h following exposure to LDR. Cell apoptosis (D) and clonogenic survival (E) of MCF-10A cell exposed to Sham, LDR, HDR or LDR 8 h before HDR. Data are represented as mean G SEM, n = 3; **p < 0.01, Student's t test.
(F) LDR-induced radioprotection was measured by apoptosis with flow cytometry in Per2 wt and Per2 def BMMNCs 24 h after HDR or LDR 8 h before HDR. Data are represented as mean G SEM, n = 6, *p < 0.05, **p < 0.01, ANOVA two-way test was applied.
(G-I) LDR-induced radioprotection was measured by GM-CFU assay in Per2 wt and Per2 def BMMNCs after HDR or LDR 8 h before HDR. Data are represented as mean G SEM, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA two-way test was applied. Cell proliferation (H) and clonogenic survival (I) of MCF-10A cells transfected with siPer2 or scrambled siPer2 following exposure to Sham, LDR, HDR or LDR 8 h before HDR. Data are represented as mean G SEM, n = 3, **p < 0.01, ns p > 0.05, ANOVA two-way test was applied.  Figure 5G). In addition, PER2, pGSK3b(S9), and active b-catenin were enhanced by LDR in GSK3b wt , but not detected in GSK3b ko MEFs (Figures 5H and 5I). The basal level of active b-catenin and Per2 were elevated in the GSK3b ko MEFs, but not enhanced by LDR, indicates that PER2-GSK3b interaction is required for LDR radioprotection ( Figures 5H and 5I). Together, these results revealed that PER2/p-GSK3bS9 complex functions as a signaling switch for b-catenin activation to upregulate LDR-induced prosurvival genes.

PER2/pGSK3b(Ser9) interaction extended pGSK3b(Ser9) life time, activating b-catenin for Per2 transcription
Active GSK3b is shown to phosphorylate and activate PER2 for nuclear translocation. 30 We assumed that PER2 may directly interact with the inactive form pGSK3b(S9) and as a result activate the b-catenin/TCFregulated prosurvival network in LDR-induced adaptive radioprotection. Indeed, although PER2/ pGSK3b(S9) complex was detected in the non-LDR control, LDR strikingly enhanced the complex formation at 12 h after LDR ( Figure 6A). To confirm the specificity of pGSK3b(S9) with interaction, we co-transfected the mutant pGSK3b S9A-HA and Per2-V5 plasmids into 293T cells. Co-immunoprecipitation identified PER2 interaction with wild-type GSK3b(S9), but not the S9A mutant in 293T cells with overexpressing Per2 ( Figures 6B and S7D). In consistence, cycloheximide (CHX) chase assay revealed that both active form of b-catenin and pGSK3b(S9) were maintained in Per2 wt BMMNCs whereas active b-catenin was faintly detectable with a rapid pGSK3b(S9) degradation in Per2 def BMMNCs ( Figures 6C and 6D), indicating that the formation of PER2/pGSK3b(S9) complex prolonged inactive form of pGSK3b(S9), enhancing active b-catenin. Furthermore, the nuclear translocated active b-catenin paralleled with reduced cytoplasmic active b-catenin in Per2 wt /GSK3b wt MEFs 12 h after LDR ( Figure 6E), indicating a PER2/pGSK3b/ b-catenin/TCF axis that upregulates Per2 transcription and a cluster of prosurvival genes. In Wnt/b-catenin signaling, the active b-catenin functions as a transcriptional coactivator for transcriptional factors T cell factor/lymphoid enhancer factor (TCF/LEF) with binding elements at the promoter region of the effector genes. 42 Consistently, database searching revealed that the Per2 promoter region contains the well-defined b-catenin-TCF/LEF domains including LEF-1/TCF-1A, TCF-4, TCF-1(P), TCF-2, and TCF-3 identified in the human and mouse Per2 promoter region ( Figure S8A Table A, S8B Table B). We then constructed luciferase reporters driven by the cloned Per2 promoter enriched with the TCF/LEF domains. Luciferase assay result showed enhanced reporter activity by LDR in GSK3b wt MEFs, but not in the GSK3b ko MEFs ( Figure 6F). Moreover, LDR-induced Per2 promoter transactivation was also inhibited by Calphostin C that blocks GSK3b phosphorylation reducing the active b-catenin; Cal, 0.1 mM, 1 h; 0.3 mM, 1 h) ( Figures 6G and S9A). b-catenin mediated Per2 expression was further evidenced by the increased b-catenin activity in LDR treated cells and reversed by b-catenin inhibition with calphostin C both in MEFs and MCF-10A cells ( Figures 6H and S9B). Thus, LDR-induced PER2/pGSK3b/b-catenin/Per2 loop is suggested to play a major role in LDR-induced adaptive radioprotection.

PER2 is related to b-catenin/TCF/LEF prosurvival effector genes
More in-depth analysis of the Per2 wt versus Per2 def transcriptome demonstrates PER2/b-catenin boosted LDR adaptive radioresistance. Activation of Wnt/b-catenin/TCF/LEF pathway is suggested iScience Article to enhance normal tissue tolerance to radiation. 43 We attempted to define the TCF/LEF effector genes differently expressed in Per2 wt versus Per2 def BMpHSCs and revealed a cluster of PER2 related TCF effector genes ( Figure 7A) indicating a short list of potential TCF regulated prosurvival genes for DDR and mitochondrial metabolism ( Figure S10). The up-regulated TCF/LEF effector genes in Per2 wt include the regulation of DNA metabolic process, regulation of myeloid cell differentiation, hematopoiesis and macromolecule synthesis ( Figure 7B). Together with other potential PER2-associated effector genes upregulated in Per2 wt versus Per2 def BMpHSCs, a prosurvival network is illustrated under upregulated LEF/TCF effector genes interaction with the cascade of upregulated DNA repair, mitochondrial functions and respiration, and lipid metabolism ( Figure 7C). Altogether, our current work reveals a PER2/pGSK3b/b-catenin/Per2 cascade that is activated for cellular adaptive radioprotection induced by exposure to low-dose radiation. Under LDR, PER2 forms complex with pGSK3b to enhance b-catenin/TCF/LEF regulated effector genes including Per2 and a cluster of prosurvival target genes coordinating with DNA repair and mitochondrial metabolism for cells to survive subsequential genotoxic stresses Figure 7D).

DISCUSSION
Natural low-dose radiation and circadian rhythm (CR) may have coordinatively contributed to the environment-adaptive capacity in cells. This study provides experimental evidence that PER2, a fundamental factor in CR functions in signaling cellular adaptive radioprotection induced by low-dose radiation. CR is welldefined to affect cellular stress response 44 such as CR-associated cell radiosensitivity 6,45,46 and CR-related proliferation and differentiation of BMpHSCs. 47 Pre-exposure of mammalian cells to LDR can generate a prosurvival advantage to subsequent genotoxic insults such as the cytotoxicity of high dose radiation. 11,12,37,48,49 Data from circulating leukocytes of humans with simulated night shift showed circadian dysregulation of DNA repair genes and elevated DNA damage 50 whereas cells exposed to LDR (10 cGy) initiated an adaptive cellular homeostasis raising their tolerance to subsequent more severe genotoxic stress. 12,51-54 We report here that in addition to CR regulation, PER2 protein can function as a signaling element in LDR-induced radioprotection. A PER2/pGSK3b/b-catenin/Per2 loop is suggested by PER2 enhanced prosurvival response.
On the animal level, no difference was observed in survival and tumor incidence between Per2 def versus Per2 wt mice following WBI with a lower radiation dose (4 Gy) which was expected to accelerate aging among the survival animals. 33 However, Fu's 7 research showed that Per2 deficient mice were enhanced sensitivity to radiation associated tumor development causing lowered animal survival in Per2 deficient mice. Under such experimental setting, a dose of 4 Gy radiation clearly enhances tumor formation in Per2 deficient mice indicating that Per2 deficiency is more sensitive to radiation-induced cancer risk, which is consistent with our data of whole-body irradiation with 7 Gy although the radiation doses and time period observed are different between the two experiments. The survival data in our current study further demonstrated a PER2-related animal survival advantage following WBI using a higher dose range 7-12 Gy ( Figure 1A). The difference between these two animal studies may be related to the degree of DNA damages and PER2-associated repair capacity in stem cells including the DNA repair ability and mitochondrial function in the bone marrow pHSCs and monocytes. iScience Article Consistent with the Per2-related survival, Per2 def cell radiosensitivity is linked with an impaired DNA repair capacity with reduced BMpHSC population and mitochondrial function ( Figures 1B-1G, 2A, 2B, and 3D-3F). These are supported by gene expression profiling generated from Per2 wt versus Per2 def BMpHSCs indicating a cluster of prosurvival genes involved in DNA repair and mitochondrial functions silenced in the Per2 def cells. Among many other potential PER2-regulated effector genes, the categories of genes involved in DNA repair and mitochondrial metabolic functions are focused based on the findings that mitochondrial dysfunction reduces the maintenance of genomic instability after radiation. 35,55 The reduced/silenced genes in DNA damage response and mitochondrial activity appear to be the major cellular deficiencies causing the lack of LDR-induced radioprotection in Per2 deficient animals.
On the cellular level, the current study reveals that Per2 wt cells exhibit adaptive radioprotection induced by LDR whereas Per2 def cells show no radioprotection and are even sensitive to LDR-induced apoptosis with lowered clonogenic survival ( Figures 2F, 2G, 4F, and 4G). In addition to DNA repair and mitochondrial homeostasis, such PER2-mediated intrinsic radioprotection may be via p53 since PER2 can directly interact with p53 to sustain a homeostatic status in genotoxic stress. 27 Although our current study could not exclude the possibility of PER2-p53 interaction, a peak PER2 protein accumulation induced by LDR is around 8-12 h, which is probably after the peak activation of p53 reported at 4 h after LDR with 10 cGy delivered within 72 h period of time. 56 Again, consistent with the finding from mouse cells, knockdown of Per2 in MCF-10A cells also impaired the LDR-induced survival advantages ( Figures 4H and 4I). Therefore, although this work could not exclude the potential functions in cell radiosensitivity in different CR period, the clock protein PER2 itself is demonstrated to induce cellular adaptive radioprotection. Adjusting Per2 gene expression without CR adjustment may be potential effective approach to reduce cell radiosensitivity such as reducing normal tissue injury in cancer patient radiotherapy.
Wnt/b-catenin signaling is a well-defined prosurvival pathway with b-catenin functioning as the transcriptional coactivator for TCF/LEF effector gene regulation in which b-catenin is regulated following phosphorylation. 57 Activation of Wnt/b-catenin signaling is indicated to prevent radiation damage to normal tissues leading to cell radioresistance 58,59 and LDR-mediated Wnt/b-Catenin activation is evidenced in the proliferation of neural stem cells. 40 In consistence with these results, our current findings further reveal a direct interaction between PER2 and pGSK3b(S9) in the Wnt/b-catenin axis. This previously unknown mechanism is shown to activate the PER2/pGSK3b/b-catenin/Per2 loop underlying PER2-promoted prosurvival advantage under genotoxic stress. Together with the defined association of PER2 with GSK3b(S9)/b-catenin via database analysis, b-catenin is reduced in the signaling pathway in Per2 deficient cells, which suggests that PER2 functions as the upstream responsory for GSK3b/b-catenin pathway for LDR-induced adaptive radioprotection.
Prominently, the PER2/pGSK3b(S9) complex may hold a fundamental biological function. An active GSK3b is shown to play a critical role in regulating pleiotropic cellular functions including the phosphorylation and nuclear transportation of PER2. 30 We showed that following LDR, PER2 interacts with the inactive form pGSK3b(S9) to boost a temporary, but significant cellular stress adaptive status via b-catenin/TCF regulated prosurvival network. Per2 def BMMNCs showed a reduced b-catenin without LDR-induced b-catenin activation ( Figures 5C and 5F), whereas LDR-induced pGSK3b(S9) was concurred with activation b-catenin and Per2 induction ( Figures 5D and 5H). The PER2/pGSK3b/b-catenin/Per2 loop is also supported by the direct interaction of PER2 and pGSK3b(S9) and diminished by mutant pGSK3b(S9A). GSK3b-KO cells unable to induce radioprotection response by LDR ( Figure 5E) and LDR enhanced reporter activity driven by mouse Per2 promoter in Per2 wt /GSK3b wt cells was dose-dependently inhibited by b-catenin inhibitor Calphostin C (Figures 6G and 6H). The PER2-Per2 feedforward activity for enhancing cell survival is also illustrated by the findings with Per2 promoter featured with multiple TCL/LEF binding sites responsible for b-catenin mediated transactivation. LEF-1 is a member of the LEF-1/T-cell-factor (TCF) family of iScience Article transcription factors, a potential transcriptional regulator for b-catenin controlled Per2 transcription since LEF-1 is regulated by Wnt pathway for maintenance of cellular genomic integrity. 60 Again, LDR-mediated Per2 promotor activation was more enhanced in WT versus GSK3b-KO cells ( Figure 6F) whereas inhibiting b-catenin also suppressed Per2 promotor activity and Per2 expression. Together, these results indicate that PER2/pGSK3b(S9) complex functions to regulate LDR-induced radioprotection by b-catenin/TCF/LEFregulated prosurvival genes ( Figure 7D).
In summary, this study provides evidence indicating that the clock protein PER2 participates in radiationinduced adaptive response via PER2/pGSK3b/b-catenin/Per2 loop that upregulate a cluster of prosurvival genes including DNA repair and mitochondrial metabolism. The PER2/pGSK3b/b-catenin/Per2 cascade may be targeted to raise normal cell tolerance to radiation-induced injury.

Limitations of the study
One limitation of this study is the uncertainty of PER2 in the radioprotection of normal human tissue.
Although several human cell lines and primary human epithelial cells are indeed induced Per2 expression with radioprotection and Per2 Def mice showed an enhanced radiosensitivity, the PER2/pGSK3b/b-catenin/ Per2 loop is not identified in vivo models. Thus, the conclusion of this work could be further validated by in vivo studies and/or pre-clinical trials with patients after receiving an equivalent diagnostic low-level irradiation. Additional studies may further elucidate if PER2/pGSK3b/b-catenin/Per2 loop is involved in a dynamic pattern in irradiated human cells regarding its function in sustaining cellular homeostasis via keeping an elevated level of prosurvival genes.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
The authors declare no competing interests.

INCLUSION AND DIVERSITY
One or more of the authors of this article self-identifies as an underrepresented ethnic minority in their field of research or within their geographical location.

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jian Jian Li (jijli@ucdavis.edu).

Materials availability
Any additional information required to reanalyse the data reported in this paper is available from the lead contact upon request. iScience Article Rad51 (Santa Cruz Biotechnology, sc-7410) and gH2AX were used. Cell were then incubated at room temperature with the fluorescence-conjugated secondary antibody for 1 h in the dark. Cell nuclei were then counterstained with DAPI contained in the mounting solution and the DNA repair foci images were acquired using a Zeiss LSM710 confocal microscope system and analyzed with ImageJ software. The experiment was repeated three times and at least 30-40 cells were scored for each sample. Data represent the percentage of cells containing more than three foci per nucleus.
RNAseq libraries were prepared from NEBNext mRNA Library Prep Master Mix Kit and NEBNext Multiplex Oligos (New England BioLabs, Ipswich, MA) according to the manufacturer's standard protocol. Briefly, poly-adenylated mRNA was purified from total RNA by two rounds of binding to oligo d(T) 25 paramagnetic beads, which was then followed by mRNA fragmentation by incubation in the presence of divalent cations at 94 C for 5 min. Double-stranded cDNA was then generated by random-primed first-strand synthesis with ProtoScript II reverse transcriptase and subsequent second strand synthesis with NEBNext Second Strand Synthesis Enzyme Mix. The cDNA was then blunt-ended and 3 0 -dA tailed by incubation with Klenow Fragment (3 0 / 5 0 exo-) and dATP. Illumina paired-end (PE) adapters were then ligated, followed by size selection and purification of the cDNA library with Agencourt AMPure XP beads. Libraries were then enriched and indexed by high-fidelity PCR amplification (12 cycles) with Q5 High-Fidelity DNA Polymerase (NEB) and adapter-specific and multiplex primers.

Next generation sequencing (NGS) data analysis
Image processing, base calling, quality scoring (Phred), and sample demultiplexing were executed by Hi-Seq Control Software with Real Time Analysis (HCS 1.5/RTA 1.13) and CASAVA 1.8 software (Illumina; San Diego, CA). RNAseq sequence reads (FASTQ format) were aligned to the reference human genome assembly (Feb. 2009, GRCh37/hg19) using TopHat software which performs splice junction mapping after read alignment with Bowtie2. Gene-and transcript-level expression were comprehensively quantified with Cufflinks software which performed 1) transcript assembly, 2) identification of splice variants, and 3) quantification of normalized expression as FPKM (fragments per kilobase of transcript per million mapped reads) values. Differentially expressed genes in Per2 wt verses Per2 def BMMNCs were determined using the Cufflinks program. Biological interpretation of the resulting gene list was performed using functional annotation and clustering tools available at the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources 6.7 with calculated enrichment scores based on a Fisher Exact Test. 64 Subsequently, network analysis of the differentially-regulated genes was performed using VisANT tool based on functional relations in the Predictome database. 65 Gene ontology biological process enrichment analysis was performed using the ggplot2 R language packages.

LDR of primary breast epithelial cells
Breast tissue biopsies from three healthy individuals with reduction mammoplasty surgeries were collected under standard procedure with consent and the mammary epithelial cells were isolated and cultured following the established protocol. 66 The primary cultured epithelial cells were pooled and passaged for 2-3 times and treated with sham or LDR (10 cGy) and cell lysate was harvested 12 h after treatment for analysis of induced Per2 expression by western blot.

Measuring clonogenic survival
Cells constantly passaged every two days to 80% confluency were exposed to LDR (10 cGy), LDR or LDR 12 h before HDR (5 Gy) and 800 cells were seeded into each well of 6-well plates. Both irradiated and control cells were cultured for 10 to 14 days and colonies were stained with Coomassie blue. The colony with more than 50 cells were counted. The colony images were obtained by Nikon microscope. The clonogenicity were calculated by the percentages of colonies formed from seeded cells in each group.

siRNA-mediated Per2 inhibition
siRNA against human PER2 gene was designed and synthesized with the Silencer siRNA Construction Kit (Ambion, Cat. 1620). The primers used to synthesize the targeted and scrambled siRNAs for PER2 were as follows: Scramble: 5-AAATATGTGCGTACCTAGCTTCCTGTCTC-3 0 , and targeted Per2: 5 0 -AATGAAGAGT ATTACCAGCTGCCTGTCTC-3 0 . Cells were seeded to achieve 30-50% confluency on the day of transfection and siRNA transfection was conducted using Lipofectamine RNAiMAX reagent (Invitrogen, Cat. 13778-100). Briefly, cells were consistently passaged 2 times before the transfection and seeded into 60-mm cell culture dish one day before the transfection with a 30-50% confluency. After overnight transfection, half amount of fresh medium was added directly into the transfection culture medium and the culture was kept for another 24 h. Scrambled RNA Duplex (Ambion, Austin, TX) served as the specificity control.

Measuring cell proliferation
MTT (Sigma, Cat. MÀ2128) was applied to monitor cell proliferation treated with LDR, HDR or LDR 12 h before HDR in WT MCF-10A cell and siPer2 transfected MCF-10A cells. Briefly, 0.8310 4 cells/well were seeded in 96-well plates and incubated for 48 h. Then, the medium was replaced with 100 mL of fresh medium containing 0.5 mg/mL MTT reagent for further 4 h incubation. The medium was removed, and the formazan crystals were solubilized by adding 100 mL of Dimethyl Sulfoxide (DMSO, Sigma, Cat. D2650) for 30 min. The absorbance of the dissolved formazan crystals was recorded using the microplate spectrophotometer (Molecular Devices) at 540-570 nm.

Co-immunoprecipitation
Proper antibody was incubated with Protein A or G magnetic beads for 1 h at RT and the protein extracts (500 mg) in 500 mL of 1x RIPA buffer were incubated with antibody coupled magnetic beads on rotator overnight at 4 C. Then, the captured proteins were eluted and denatured by loading buffer with boiling at 95 C for 10 min. The supernatant was applied to SDS-PAGE that was followed by western blotting. The antibodies applied in co-IP were anti-Per2 antibody (Cat. 611138), anti-pGSK3b (Santa Cruz; Cat. 5558T), V5-tag antibody (Proteintech; Cat. 66007-1), p-serine (Cat. 05-1000X) and p-threonine (Cat. SAB5600203).

Transfection of Per2-expressing plasmids
Human Per2 cDNA vector pLenti6.3/V5-DEST was kindly provided by Dr. Min Gyu Lee from The University of Texas MD Anderson Cancer Center. Lentiviral particles were packaged in HEK293T cells according to the protocol from Addgene. The stable Per2 overexpression 293T (Per2-OE) was constructed by infecting 293T cell with packaged lentiviruses according to published protocol. GSK3b wt and GSK3b S9A-mut plasmids were derived from Addgene and transfected with the Per2-OE 293T cells following the protocol of turbo reagent transfection (Thermo Scientific, Cat. R0531). The transfected cells were cultured for 24 h before LDR (10 cGy) and cells were collected 12 h after LDR for immunoprecipitation. To measure the basal and LDR-induced transcription factors, we utilized plasmid containing the Per2 promoter region (pGL3-basic-mPer2) and included a b-catenin predicted binding site. Cells were co-transfected with pGL2-basic-Per2-2 and Renilla Luciferase-Pol.III by turbo reagent for 24 h then treated cells with LDR. Luciferase activity was measured 12 h after LDR by using 20 mL of total cell lysates and 100 mL of luciferase assay reagent (Promega, Cat. E1483) as described previously [12,36]. Equal amount of cell lysates was used for the assessment of Renilla activity to normalize luciferase activity.

Protein degradation analysis
Chase assay was performed to determine the stability of pGSK3b and active b-catenin in Per2 wt and Per2 def mouse BMMNCs treated with LDR. Briefly, 5310 6 cells were seeded in 100 mm plate and incubated for 24 h before receiving LDR treatment. After 12 h, the medium was replaced with 3 mL of fresh medium containing 30 mg/mL cycloheximide for further incubation. The cells were collected at different time points (0 h, 1 h, 2 h, 4 h) and total cell protein was quantified by BCA Protein Assay kit and western blots performed.

Nuclear and cytosolic Per2 and b-catenin level
Western blot was used to investigate Per2 and b-catenin in nuclear and in cytosolic fractions. Cells were digested using lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT, 1% IGEPAL 630) with protease inhibitor cocktail (Sigma, Cat. P8340). Cells were incubated for 7 min on shaker at 4 C and transferred to 1.5 mL centrifuge tube, followed by centrifuging at 12,000 rpm for 1 min. The supernatant obtained correspond to the cytosolic fraction and the pellet was washed by washing buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT). The pellet was then resuspended in extraction buffer (10 mM HEPES, 410 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT, 0.2 mM EDTA, and 25% Glycerol). The supernatant was saved as nuclear fraction after 14,000 rpm centrifugation at 4 C for 10 min. Total protein content was measured by BCA Protein Assay kit and western blot was performed.

Statistical analyses
All statistical analyses were using GraphPad Prism 8.0. Software (GraphPad Software, Inc., San Diego, CA). Data were presented as mean G SEM For comparison of survival curves, a log rank test was applied. Other results were analyzed by the two-tailed student t-test and one way ANOVA with data considered significant at p <0.05. All data were presented as the standard error of the mean with at least three individual experiments.